검안및콘택트렌즈학회지 2018 년제 17 권제 4 호 Ann Optom Contact Lens 2018;17(4):89-96 ISSN 2384-0919 (Print) ISSN 2384-0927 (Online) Review Article 안과영역에서의차세대염기서열분석 Next-generation Sequencing in Inherited Eye Diseases 한진우 Jinu Han, MD 연세대학교의과대학강남세브란스병원안과학교실시기능개발연구소 Institute of Vision Research, Department of Ophthalmology, Gangnam Severance Hospital, Yonsei University of College of Medicine, Seoul, Korea Next-generation sequencing is widely used in inherited diseases and cancer genetics fields. Next-generation sequencing technology provides accurate diagnosis in genetically heterogeneous disorders such as retinitis pigmentosa, Leber congenital amaurosis, or cone-rod dystrophy. However, the precise interpretation of variants produced by massively parallel sequencing is somewhat difficult to most of ophthalmologists, and misinterpretation of these variants lead to unwanted devastating consequences to the patients and their family. The molecular genetic findings need to be carefully evaluated in the context of the clinical findings to avoid misdiagnosis. Gene therapy trials are already in the market for specific forms of Leber congenital amaurosis. We are in the middle of exiting era of effective treatment for patients with inherited eye diseases, which was considered as incurable in the past. To success such a treatment, molecular diagnosis will become essential. Ann Optom Contact Lens 2018;17(4):89-96 Key Words: Inherited eye disease; Inherited retinal disease; Next-generation sequencing 서 론 1977년대 Sanger et al 1 에의해개발된 DNA 염기서열분석기술은이후 2007년경차세대염기서열분석 (next-generation sequencing) 으로이어져현대의학발전에많은기여를하고있다. 차세대염기서열분석은병렬적 (massively parallel) 으로십억이상의리드 (reads, 염기서열단편 ) 를처리하여, 염기서열분석에걸리는시간및비용을획기적으로줄여주었으며, 이에따라유전체연구의효과적인방법 Received: 2018. 12. 5. Revised: 2018. 12. 18. Accepted: 2018. 12. 18. Address reprint requests to Jinu Han, MD Institute of Vision Research, Department of Ophthalmology, Gangnam Severance Hospital, #211 Eonju-ro, Gangnam-gu, Seoul 06273, Korea Tel: 82-2-2019-3445, Fax: 82-2-3463-1049 E-mail: jinuhan@yuhs.ac * This research was supported by a fund (2018-ER6902-00) by Research of Korea Centers for Disease Control and Prevention. 으로자리잡고있다. 이러한 DNA 염기서열분석기술이단백질을전사하는부위인 exon과 exon-intron의경계만염기서열분석하는 exome sequencing, 혹은 intron을포함한전체염기서열을분석하는 genome sequencing이상대적으로적은비용에가능하게되었으며종양의조직에서돌연변이를찾는종양유전체학그리고유전적으로다양한유전자에의해질환이발생하는 (genetically heterogeneous) 유전질환의진단등에현재이용이되고있다. 안과적인질환은한개의유전자가그돌연변이의위치에따라다양한표현형을나타내기도하고 (Fig. 1), 여러개의서로다른유전자의변이가유사한표현형을보이는경우도많다. 안과의분야에서 Leber congenital amaurosis, rod-cone dystrophy, congenital cataract 등의유전질환에서원인돌연변이를찾는데이용되고있으며, 이렇게유전학적으로여러개의유전자가병을일으키는유전질환에서진단율을획기적으로높였으며, 현재여러유전성안질환에서의진단율은약 50-75% 정도로보고가되고있다. 2-4 또한, uveal melanoma Copyright 2018, The Korean Optometry Society The Korean Contact Lens Study Society Annals of Optometry and Contact Lens is an Open Access Journal. All articles are distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 89
- 검안및콘택트렌즈학회지 2018 년제 17 권제 4 호 - A B C D Figure 1. Leber congenital amaurosis and rod-cone dystrophy phenotypes according to different SPATA7 mutations. (A, B) Fundus photograph showed marbled fundus, and optical coherence tomography revealed degeneration of photoreceptor layer with minimal remaining at the fovea. (C, D) Wide fundus photographs showed pigmentary retinopathy and optical coherence tomography revealed that photoreceptor layer was remained in the central fovea. 와같은안종양학에도차세대염기서열분석이이용이되고있으며, 5 최근에는 ribonucleic acid (RNA)-seq과 single cell sequencing 기법으로 cell-level 단위에서의 expression 을볼수있는방법이등장하여질병의매커니즘 (mechanism) 규명에다양하게응용이되고있다. 6 2015년미국에서 Precision Medicine Initiative 프로젝트가시작되면서백만명의환자들의유전체정보를얻어환자개개인에맞는맞춤치료를제공하는것을목표로하고있으며, 그에따라환자개개인별로그에해당되는임상검사와적절한약이제공되는것이요구되는시대일것으로생각된다. 7,8 그러므로차세대염기서열분석은현대의학의필수적인부분으로자리잡고있으나아직이부분에대한안과의사들의이해가부족한실정이다. 따라서, 차세대염기서열분석에대한기초적인이해가필요할것으로생각되며, next generation sequencing (NGS) 방법의장단점과한계, 변이해석에있어유의할점이어떠한것이있는지자세하게알아볼필요가있을것이다. 본고에서는 NGS 데이터가원시데이터에서시작하여유전변이추출에이르는단계들의기본적인개념을소개하고, 안과질환에서의적용, NGS 방법의한계점및변이의해석을어떻게할것인지, 안과질환에서어떻게응용을할수있을지에대하여살펴보고자한다. 하는 targeted panel 염기서열분석은임상적으로현재적용이가능한분야이며, retinitis pigmentsoa, congenital hearing loss, Charcot- Marie-Tooth병에서보험적용이되고있으며, benchtop 시퀀서인 MiSeq, NextSeq550으로가능하다. 하지만 whole exome 혹은 whole genome 염기서열분석은생산되는정보의양이많으므로대개는 HiSeq 혹은 NovaSeq 을이용하여염기서열분석을하게된다. MiSeq과 HiSeq은 4-Dye system으로네종류의염기마다서로다른형광을사용하지만 NextSeq550과 NovaSeq은 C (cytosine) 은빨강, T (thymine) 은초록, A (adenine) 는두색깔의혼합그리고 G (guanine) 는형광이없는형태로표지한다. 사용되는형광의개수를절반으로줄임으로서염기서열분석원가를절감할수있으며, 데이터의 quality도기존의제품과유사하다고보고되고있다. Long reads 시퀀서인 PacBio 와 Oxford Nano는아직까지는 error rate가높다고여겨져환자의임상적인유전진단에널리사용되고있지는않다. 따라서, 각각연구나임상목적에맞는염기서열분석기계를선택하면될것으로생각된다 (Table 1). NGS 데이터의 data pre-processing 및 SNP/INDEL calling Sequencing 장비의선택 차세대염기서열분석의업계선두는단연 illumina이다. MiSeq, NextSeq550, HiSeq 등을비롯하여최근에출시가된 NovaSeq으로염기서열분석비용이획기적으로낮아졌다. Targeted panel sequencing, whole exome 혹은 whole genome에따라장비를선택해서염기서열분석을하면가장낮은비용으로원하는 throughput을얻을수있다. 대개약 1,000개미만의원하는유전자를구성하여염기서열분석 NGS의여러데이터를분석하고논문을이해하기위해서는여러가지용어들을우선알아둘필요가있다 (Table 2). NGS 염기서열분석방법을간단히설명하면환자에게서추출된 DNA는초음파분쇄기에의해 300-700 bp의작은조각들로잘라지며, 분쇄된 DNA를 adapter sequence에붙여라이브러리를생성한다. 이 adapter에붙은작은 DNA 조각들이 flow cell에서클러스터를형성하며증폭과정을통하여보통 100-150개정도의염기로구성된짧은서열조각인리드 (reads) 를생성하게되며이러한리드들의정보 90
- 한진우 : 안과에서의차세대염기서열분석 - Table 1. Technical specification of sequencing platform Sequencing platform Total output (bases per run) Total reads (million per run) Read length (bases) Run time (days) Purpose Illumina MiSeq 300 Mb -15 Gb 50 M 2 300 bp 0.2-2.7 Targeted panel or amplicon sequencing NextSeq 550 25-120 Gb 260 M-800 M 2 75 bp 1.5-3 Midthroughput to highthroughput versatile use HiSeq X 1.6-1.8 Tb 6,000 M 2 150 bp <3 Only for large scale whole genome sequencing NovaSeq 0.2-3 Tb 1,300-20,000 M 2 150 bp <2 Scalable platform, flexible performance ThermoFisher Ion Torrent 0.3-50 Gb 3-130 M 2 200 bp 0.2-1 Diverse sequencing data with as little as 10ng sample PacBio Sequel system 500 Mb-16 Gb 55-880 M Up to 60 Kb <0.1-0.3 Useful in the studies of de novo assembly Long reads sequecning Nanopore MinION Up to 42 Gb Up to 4.4 M 230-300 kb 2 Portable sequencing instrument Mb = mega base; Gb = giga base; M = million; bp = base pair; Tb = tera base; Kb = kilo base. Table 2. Terms used in next-generation sequencing Term Explanations BAM SAM의 binary version인 BAM 파일이며, SAM이아닌 BAM 파일로저장하면용량을줄일수있음 Coverage Sample DNA에서평균혹은각각의 base에 align 되는중복되지않은다른리드의숫자 ( 혹은평균갯수 ) FASTA/FASTQ FASTA 란 nucleic acid sequence나 protein sequence를저장하는 format을말하며, FASTQ는 nucleotide sequence 정보와그에해당하는 quality score가같이있는저장파일을말함 GATK Genome Analysis ToolKit의약자로 broad institute에서제공하는 SNP/indel calling 시사용하는표준 bioinformatics analysis tool임 SAM Sequence alignment/map 포맷으로서 nucleotide sequence 정보와 reference sequence에정렬되어있는정보가함께있는파일포맷을말함 Throughput 염기서열분석머신으로생산되는 base의양을말하며데이터의크기를뜻함 ( 예, 7Gb으로 whole exome sequencing 정보를생산했다고하면, 7 Giga base의정보가생산되었다는뜻 ) VCF Variant call format의약자로해당샘플에서 reference genome에대비하여어떤변이정보를가지고있는지나타내는파일포맷 BAM = binary alignment map; SAM = sequence alignment map; GATK = Genome Analysis Toolkit ; SNP = single nucleotide polymorphism; VCF = variant caller format. 가있는파일을 FASTQ 형식의파일로저장하게된다. 수많은 DNA 단편조각인리드들을 burrows-wheeler aligner 를통하여참조유전체 (reference genome) 에리드를정렬 (alignment/mapping) 하게되고, 9 중복되는리드를표시하는작업을거치고 (mark duplicate), mismatch되는부분의 indel realignment와 base quality score에따라 recalibration 과정을거쳐분석에이용될수있는 BAM 파일을생성하게된다. 분석에이용될수있는 BAM 파일에서 reference genome과는다른흔하지않은변이가있는부분을베이지안분석과 local de novo assembly 등의과정을거치는 haplotypecaller를통하여변이가존재하는부분에대하여보다정확한통계방법으로 genotype에대한정보를생산한다. 10 이러한분석과정은 Genome Analysis ToolKit의 best practice가현재표준 분석방법으로쓰이고있다. 10 또한, 이렇게생성된 variant call format file을여러명의 sample들을합쳐서분석함으로써 false-positive와염기서열분석에러부분들을필터링하는후처리과정을거쳐분석한유전체에서어떠한부분에변이가있으며이부분의변이가 heterozygote인지 homozygote 상태인지결정이된다. Variant annotation/filtering 이렇게여러가지의처리과정을거쳐나온변이들이어떤기능을하는변이들인지표지하는과정을 variant annotation이라고한다. 한사람의 exome sequencing 결과로약 2-3만개의변이가나오게되는데이러한변이들이 synon- 91
- 검안및콘택트렌즈학회지 2018 년제 17 권제 4 호 - ymous, missense, nonsense, splice site, 혹은 intronic 변이인지표지를하고, 일반인집단에서이러한변이가흔하게발견되는변이인지드문변이인지 minor allele frequency를통하여확인한다. 1000 Genomes Project, ESP6500, ExAC, gnomad 등의많은대규모의유전체연구에서일반인집단에서의변이의 minor allele frequency를확인할수있으며, 특히이런 minor allele frequency를확인할때같은인종에서해당변이의 minor allele frequency를확인하는것이중요하다 (Fig. 2). 예를들어어떠한변이를관찰하였는데해당질환이매우드문질환이고우성유전하는질환이라면정상집단에서해당변이가없거나아주드문경우여 야할것이다. 여기서고려되어야할부분이병의평균적인발현시기, 변이를가지고있을때얼마나표현형이나타나는지 (penetrance), 해당질환이얼마나드문병인지등을종합적으로고려하여판단한다. 흔히쓰는 database로 gnomad browser와 ExAC Browser가있으며, 한국인 1,722명의유전체에존재하는한국인참조유전체데이터베이스정보가공개되어있다 (Table 3). 또한, 이러한변이가여러다른종간에서잘보존되어있는지, 그리고변이가단백질의기능에어떠한영향을미치는지예측하는여러가지도구들로표지를하게된다 (Table 4). PolyPhen2와 SIFT 스코어는 missense 변이만 prediction할수있으며, 최근에는 functional Figure 2. Minor allele frequency of NMNAT1 c.709c>t variant in gnome aggregation dataset (gnomad) browser. The web browser displayed minor allele frequency in different ethnic population. Table 3. Population, disease-specific, and sequence databases Type of database Name of database Website Population database Exome aggregation consortium http://exac.broadinstitute.org/ GnomAD browser http://gnomad.broadinstitute.org/ Exome server project http://evs.gs.washington.edu/evs/ Korean reference Genome database http://152.99.75.168/krgdb Inherited disease database ClinVar https://www.ncbi.nlm.nih.gov/clinvar/ HGMD Professional https://portal.biobase-international.com/cgi-bin/portal/logi n.cgi?redirect_url=/hgmd/pro/start.php? Leiden open variation database http://www.lovd.nl/3.0/home OMIM http://www.omim.org HGMD = human gene mutation database; OMIM = online mendelian inheritance in man. 92
- 한진우 : 안과에서의차세대염기서열분석 - analysis through hidden Markov models (FATHMM) 혹은 combined annotation dependent depletion (CADD) score가 missense 변이외의 nonsense 변이와 regulatory 변이등도모두 scoring이가능하여 pathogenicity를판단할때유용하게쓰인다. 11,12 하지만이러한 in silico (=computational) 도구들에의한변이의예측은여러가지의도구를쓰지만, 그자체로강력한 pathogenicity의증거가되는것은아니며단지하나의요소로참조하여결정한다. Interpretation of variants American College of Medical Genetics Standard and Guideline 에서는변이들을분류하고해석할때 5가지의 pathogenic, likely pathogenic, uncertain significance, likely benign, benign으로분류하는가이드라인을제시하고있다. 13 이가이드라인은변이해석에있어표준지침으로자리잡고있으며, NGS 변이를해석하고리포트를받는임상의들은반드시알아야할부분이며, 해당논문을정독하여숙지할필요가있다. 어떤변이가있을때해당질환과연관되어있다고보고가된변이인지 null variant인지, 인구집단에서얼마나흔하게관찰되는변이인지등에따라해당변이가큰의미가없는 benign으로분류를할것인지 pathogenicity를가지는지점수를매기게되며각각의변이를 pathogenic인지아닌지판단하는데많은시간이걸리게된다. InterVar라는프로그램으로이런변이들에대하여자동으로 classification 을해주기도하나결국수작업으로확인해야되는부분이 존재한다 (http://wintervar.wglab.org). 14 이러한방법으로도많은변이들이 uncertain significance로남게되며이러한변이들을임상적으로해석하는데주의를요하게되며, 몇몇의경우변이의잘못된해석으로법정소송까지이어지는경우도있음을알아야한다. 15,16 또한, NGS 방법으로는상염색체열성유전하는경우 2개의변이가 cis로존재하는지 trans로존재하는지확인할수없다. 왜냐하면대부분의 2개의변이가서로다른 exon에존재하는경우가많고 NGS는 100-150 bp의짧은염기서열의조각들을단편으로환자의 genotype을추정하는것이기때문이다. 따라서, 많은경우에서가족검사를하여유사한표현형을가진가족에서같은변이가발견되는지혹은표현형이없는가족에서병적이라고간주했던변이가나오지않는지확인하여야하며, 상염색체열성유전하는질환의경우부모나자녀검사로두개의변이가 cis로존재하는지 trans로존재하는지확인을해야된다. 드물게같은 exon에 100-150 bp 내에 2 개의변이가존재하는경우도있는데이러한경우 integrative genomics viewer로확인하면서로다른리드에두개의변이가있는지확인하여상염색체열성유전하는질환에서 2개의변이가 cis로존재하는지 trans로존재하는지확인할수있다 (Fig. 3). 15 자세한표현형의기록및조사 유전성안질환의유전자검사를함에앞서환자의증상및다른전신적인동반질환에대한조사를철저히하는것 Table 4. In silico prediction algorithms Category Name Website Basis Missense prediction SIFT http://sift.jcvi.org Evolutionary conservation PolyPhen-2 http://genetics.bwh.harvard.edu/pph2 Protein structure/function and evolutionary conservation Any substitution FATHMM http://fathmm.biocompute.org.uk Evolutionary conservation Indel FATHMM-Indel http://indels.biocompute.org.uk All including indel CADD http://cadd.gs.washington.edu Contrasts annotations of fixed/nearly fixed derived alleles in humans with simulated variants Splice site prediction Human Splicing Finder http://www.umd.be/hsf/ Position-dependent logic MaxEntScan http://genes.mit.edu/burgelab/maxent/xmax Maximum entropy principle entscan_ scoreseq.html NNSplice http://www.fruitfly.org/seq_tools/splice.html Neural networks Nucleotide conservation prediction GERP http://mendel.stanford.edu/sidowlab/downlo Genomic evolutionary rate profiling ads/gerp/ index.html PhyloP http://compgen.bscb.cornell.edu/phast/ Conservation scoring and identification of conserved elements SIFT = sorting intolerant from tolerant; FATHMM = functional analysis through hidden Markov models; CADD = combined annotation dependent depletion; GERP = genomic evolutionary rate profiling. 93
- 검안및콘택트렌즈학회지 2018 년제 17 권제 4 호 - 이중요하다. 16 안과적으로는망막전위도, 안저사진, 눈떨림유무, 시야검사, 전안부이상등을철저하게조사해야되며동반된신장이상이나청력소실, 발달지연등이없는지확 인하고해당과에의뢰하도록한다. 물어보지않으면환자나보호자대부분자발적으로이야기하지않으므로안과적증상외에도다른여러가지동반된전신질환들을확인하 Figure 3. CRB1 c.1576c>t:p.arg526ter and c.1577g>c:p.arg526pro variants were presented in different reads, visualized by Integrative genomics viewer. These 2 variants were present in trans, so segregation analysis is not needed. bp = base pair. A B Figure 4. A 2-year-old girl presented at our clinic for bilateral congenital cataract. Targeted next-generation sequencing revealed NHS c.1117c>t:p.arg373ter nonsense mutation. Nance-Horan syndrome (NHS) is inherited as X-linked dominant trait. (A, B) A mutation in NHS gene causes congenital cataract and Hutchinsonian teeth. 94
- 한진우 : 안과에서의차세대염기서열분석 - 는것이필수적이다. 왜냐하면많은경우에있어서안과적질환외에도발달지연등의전신적인증상이동반되는경우가있으며이런전신적인증상을잘확인해두어야병적인변이에해당되는표현형이있는지확인해볼수가있기때문이다 (Fig. 4). 만약표현형의기록이부족하였으면유전자검사결과가나온후다시철저하게의심되는유전변이에해당되는표현형을확인하도록한다. 안과에서의적용 가장흔하게안과에서적용할수있는분야가 retinitis pigmentosa, Leber congenital amaurosis 등의유전성망막질환분야이다. 진단율은 Leber congenital amaurosis의경우약 80% 정도이며, 17,18 양안선천백내장의경우도진단율이약 70-80% 로알려져있다. 3 양안선천백내장의경우동반된전신질환에대한정보와식단조절로병의진행을늦출수있는부분이있기때문에 NGS가환자의진료에큰도움이될것으로생각된다. 19 영아안진 (infantile nystagmus) 환자도안진을일으키는질환이매우다양하여 NGS로분자유전진단이선행된다면, 필요한환자에게서만자가공명영상검사를시행할수있는등의전체적인진료비를절감 할수있는기회를제공할수있을것으로생각된다. 4 또한선천녹내장과 unexplained optic neuropathy에서도적용해볼수있을것이다. 선천녹내장의경우는 NGS 유전진단율이약 30-40% 정도로알려져있으며, 특징적인 Leber hereditary optic neuropathy나 dominant optic atrophy가아닌 optic neuropathy의경우 NGS를적용하면 atypical Wolfram syndrome 이나 Bosch-Boonstra-Schaaf optic atrophy syndrome 등진단이어렵고드문질환을정확히진단할수도있을것으로생각한다. NGS 의한계점 NGS의방법은대개 100-150 bp의짧은염기서열로이루어진수많은리드들에서정보를얻으며 polymerase chain reaction (PCR) 증폭과정을거친다. 또한 targeted panel 염기서열분석과 exome sequencing의경우 exon과 exon-intron boundary만분석이되기때문에 deep intronic 변이와 regulatory 변이는검출이안된다. Hybridization capture와 PCR 증폭과정을거치게되기때문에 GC-rich region에대한 coverage가떨어지며, 반복되는염기서열이 100-150 bp 이상으로길게존재하는경우에해당지역에대한 mapping Figure 5. Poor coverage and low quality mapping in RPGR exon 15 due to low-complex repetitive sequence. No coverage with SureSelect V6 exome on RPGR exon 15 (red arrows). IDT = integated DNA technologies; bp = base pair. 95
- 검안및콘택트렌즈학회지 2018 년제 17 권제 4 호 - quality가떨어지게된다. 예로 X-linked recessive retinitis pigmentosa의원인유전자인 RPGR 유전자의 exon 15에이러한부위가존재하는데이부위는 mutation hotspot으로해당부위의염기서열분석은 NGS 방법으로는정확하게염기서열분석하기어렵다 (Fig. 5). 20 또한, segmental duplication이존재하는부위도정확한염기서열해독이어렵다. 또한, 세개의염기서열이반복증폭되는 triplet expansion 이원인이되는 Fuch s endothelial dystrophy (CTG expansion in TCF4 gene), spinocerebellar ataxia type 7 (CAG expansion in ATXN7 gene) 의경우 NGS로정확한진단이어렵게된다는점을유념에둘필요가있다. 21,22 따라서, 이러한환자에서는기존의고식적인 NGS 방법을이용하면진단을놓치게된다는사실을알아야한다. 마지막으로, 선천성안질환및유전성안질환의대부분은진단이임상적검사만으로충분한경우가많고, 유전진단이치료로이어지지않는한계점이있다. 결 론 차세대염기서열분석이상용화된지 10년이지났지만아직도국내의안과에서소수의임상의만유전진단및연구에적용하고있으며원시데이터분석의기초에대하여무지한실정이다. 본리뷰가안과임상의가 NGS 데이터와논문을접할때조금더이해가쉬울수있도록도움이될것으로생각한다. 임상적으로안과질환에대한패널을구성할때는해당질환예를들어시신경, 망막, 전안부등으로나누어서구성을할수도있으나전체를묶어서구성하는것도좋은방법이될것으로생각한다. 왜냐하면하나의유전자가다양한안과적표현형을나타내는경우가많고, 유전자의개수가많아지면 off-target analysis로여러다른 copy number variation 등을같이확인할수있기때문이다. 유전성안질환에서차세대염기서열분석은정확한유전진단을가능하게하였으나아직까지는소수의환자에서만치료방침이달라지고대부분의환자에서는시력예후, 가족상담정도만할수있는실정이다. 하지만희귀질환의분자유전학적인매커니즘이차차밝혀짐에따라많은환자에게개개인의맞춤치료가가능하게될미래가올것으로기대한다. REFERENCES 1) Sanger F, Air GM, Barrell BG, et al. Nucleotide sequence of bacteriophage phi X174 DNA. Nature 1977;265:687-95. 2) Chaitankar V, Karakülah G, Ratnapriya R, et al. Next generation sequencing technology and genomewide data analysis: perspectives for retinal research. Prog Retin Eye Res 2016;55:1-31. 3) Gillespie RL, O'Sullivan J, Ashworth J, et al. Personalized diagnosis and management of congenital cataract by next-generation sequencing. Ophthalmology 2014;121:2124-37.e1-2. 4) Rim JH, Lee ST, Gee HY, et al. Accuracy of next-generation sequencing for molecular diagnosis in patients with infantile nystagmus syndrome. JAMA Ophthalmol 2017;135:1376-85. 5) Luscan A, Just PA, Briand A, et al. Uveal melanoma hepatic metastases mutation spectrum analysis using targeted next-generation sequencing of 400 cancer genes. Br J Ophthalmol 2015;99:437-9. 6) Grün D, van Oudenaarden A. Design and analysis of single-cell sequencing experiments. Cell 2015;163:799-810. 7) Sankar PL, Parker LS. The Precision Medicine Initiative's All of Us Research Program: an agenda for research on its ethical, legal, and social issues. Genet Med 2017;19:743-50. 8) Ashley EA. Towards precision medicine. Nat Rev Genet 2016;17: 507-22. 9) Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009;25:1754-60. 10) McKenna A, Hanna M, Banks E, et al. The Genome Analysis Toolkit: a mapreduce framework for analyzing next-generation DNA sequencing data. Genome Res 2010;20:1297-303. 11) Shihab HA, Gough J, Cooper DN, et al. Predicting the functional, molecular, and phenotypic consequences of amino acid substitutions using hidden Markov models. Hum Mutat 2013;34:57-65. 12) Kircher M, Witten DM, Jain P, et al. A general framework for estimating the relative pathogenicity of human genetic variants. Nat Genet 2014;46:310-5. 13) Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015;17:405-24. 14) Li Q, Wang K. InterVar: Clinical Interpretation of Genetic Variants by the 2015 ACMG-AMP Guidelines. Am J Hum Genet 2017;100:267-80. 15) Robinson JT, Thorvaldsdóttir H, Winckler W, et al. Integrative genomics viewer. Nat Biotechnol 2011;29:24-6. 16) Wiggs JL. Progress in diagnostic genetic testing for inherited eye disease. JAMA Ophthalmol 2017;135:1385-6. 17) Thompson JA, De Roach JN, McLaren TL, et al. The genetic profile of leber congenital amaurosis in an Australian cohort. Mol Genet Genomic Med 2017;5:652-67. 18) Han J, Rim JH, Hwang IS, et al. Diagnostic application of clinical exome sequencing in Leber congenital amaurosis. Mol Vis 2017; 23:649-59. 19) Gillespie RL, Urquhart J, Anderson B, et al. Next-generation sequencing in the diagnosis of metabolic disease marked by pediatric cataract. Ophthalmology 2016;123:217-20. 20) Stone EM, Andorf JL, Whitmore SS, et al. Clinically focused molecular investigation of 1000 consecutive families with inherited retinal disease. Ophthalmology 2017;124:1314-31. 21) Soliman AZ, Xing C, Radwan SH, et al. Correlation of severity of fuchs endothelial corneal dystrophy with triplet repeat expansion in TCF4. JAMA Ophthalmol 2015;133:1386-91. 22) Wallace SE, Bird TD. Molecular genetic testing for hereditary ataxia: what every neurologist should know. Neurol Clin Pract 2018;8:27-32. 96